Antibiotic resistant bacterial infections kill more Americans each year than colon, prostate and ovarian cancer combined. Conjugative DNA transfer generates most of the antibiotic resistant strains of bacteria that infect humans. We have recently shown that the relaxase enzyme essential to this DNA transfer process can be inhibited with nanomolar efficacy using a variety of small molecules, including some osteoporosis drugs. Relaxase inhibition prevents DNA transfer and selectively kills antibiotic resistant bacteria. The relaxase of the conjugative F plasmid is part of the large multifunctional TraI protein that also contains highly efficient helicase and putative protein-binding C-terminal domains. The relaxase, helicase and C-terminal regions of TraI are all essential for conjugative DNA transfer. This application focuses on extending our preliminary structural and chemical biology studies with the goals of understanding the molecular basis of DNA transfer and developing small molecules capable of killing antibiotic resistant bacteria. This project will accomplish four specific aims: 1. Elucidate crystal structures of a range of relaxase-inhibitor complexes. 2. Discover and synthesize new relaxase inhibitors and test their impact on conjugation and bacterial survival. 3. Unravel the role the TraI C-terminal domain plays in conjugative transfer. 4. Examine the structure, function and inhibition of the TraI conjugative helicase region. Results from these studies will provide detailed mechanistic insights into one of the first DNA manipulation systems discovered. In addition, because conserved relaxases are present in a range of pathogenic microbes, our results may provide a novel method to target the most dangerous infectious bacteria - those that are antibiotic resistant and are capable of spreading their resistance to neighboring cells. PUBLIC HEALTH RELEVANCE: Conjugative DNA transfer, the primary route by which antibiotic resistance genes spread through bacterial populations, is initiated and driven by DNA relaxase and helicase enzymes. We have recently shown that conjugative relaxases can be inhibited with nanomolar efficacy, and that this inhibition prevents DNA conjugation and selectively kills antibiotic resistant bacteria. This project will extend our preliminary structural and chemical biology discoveries with the goal of understanding the molecular basis of DNA transfer and developing drugs that potently kill antibiotic resistant bacteria.